Quantitative measurement of gene expression from fixed paraffin embedded tissue

ABSTRACT

Methods are disclosed for rapid, reliable and simple isolation of RNA, DNA and proteins from formalin-fixed paraffin-embedded tissue samples. RNA purified in this manner can be used to monitor gene expression levels. The tissue sample can be a tumor or other pathological tissue.

CROSS REFERENCE TO RELATED APPLICATION DATA

This application is a continuation of U.S. application Ser. No.10/616,203, filed Jul. 8, 2003, now pending, which is a continuation ofU.S. application Ser. No. 09/797,163, filed Mar. 1, 2001, now U.S. Pat.No. 6,610,488, which is a continuation of U.S. application Ser. No.09/469,338, filed Dec. 20, 1999, now U.S. Pat. No. 6,248,535, all ofwhich are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

The government has certain rights in this invention pursuant to grantnumber R01 CA 71716 from the National Cancer Institute of the NationalInstitutes of Health.

FIELD OF THE INVENTION

This invention relates to the field of purification of RNA, DNA andproteins from biological tissue samples.

BACKGROUND

The determination of gene expression levels in tissues is of greatimportance for accurately diagnosing human disease and is increasinglyused to determine a patient's course of treatment. Pharmacogenomicmethods can identify patients likely to respond to a particular drug andcan lead the way to new therapeutic approaches.

For example, thymidylate synthase (TS) is an integral enzyme in DNAbiosynthesis where it catalyzes the reductive methylation ofdeoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP)and provides the only route for de novo synthesis of pyrimidinenucleotides within the cell (Johnston et al., 1995). Thymidylatesynthase is a target for chemotherapeutic drugs, most commonly theantifolate agent 5-fluorouracil (5-FU). As the most effective singleagent for the treatment of colon, head and neck and breast cancers, theprimary action of 5-FU is to inhibit TS activity, resulting in depletionof intracellular thymine levels and subsequently leading to cell death.

Considerable variation in TS expression has been reported among clinicaltumor specimens from both primary tumors (Johnston et al., 1995; Lenz etal., 1995) and metastases (Farrugia et al., 1997; Leichmann et al.,1997). In colorectal cancer, for example, the ratio of TS expression intumor tissue relative to normal gastrointestinal mucosal tissue hasranged from 2 to 10 (Ardalan and Zang, 1996).

Thymidylate synthase is also known to have clinical importance in thedevelopment of tumor resistance, as demonstrated by studies that haveshown acute induction of TS protein and an increase in TS enzyme levelsin neoplastic cells after exposure to 5-FU (Spears et al. 1982; Swain etal. 1989). The ability of a tumor to acutely overexpress TS in responseto cytotoxic agents such as 5-FU may play a role in the development offluorouracil resistance. Previous studies have shown that the levels ofTS protein directly correlate with the effectiveness of 5-FU therapy,that there is a direct correlation between protein and RNA expression(Jackman et al., 1985) and that TS expression is a powerful prognosticmarker in colorectal and breast cancer (Jackman et al., 1985; Horikoshiet al., 1992).

In advanced metastatic disease, both high TS mRNA, quantified by RT-PCR,and high TS protein expression, have been shown to predict a poorresponse to fluoropyrimidine-based therapy for colorectal (Johnston etal., 1995, Farrugia et al., 1997, Leichman et al., 1997), gastric (Lenzet al., 1995, Alexander et al., 1995), and head and neck (Johnston etal., 1997) cancers. A considerable overlap between responders andnon-responders was often present in the low TS category, but patientswith TS levels above the median were predominantly non-responders. Thepredictive value of TS overexpression may be further enhanced ifcombined with other molecular characteristics such as levels ofdihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP)expression, replication error positive (RER+) status (Kitchens andBerger 1997), and p53 status (Lenz et al., 1997). Studies to date thathave evaluated the expression of TS in human tumors suggest that theability to predict response and outcome based upon TS expression inhuman tumors may provide the opportunity in the future to selectpatients most likely to benefit from TS-directed therapy.

Until now, quantitative tissue gene expression studies including thoseof TS expression have been limited to reverse transcriptase polymerasechain reaction (RT-PCR) amplification of RNA from frozen tissue.However, most pathological samples are not prepared as frozen tissues,but are routinely formalin-fixed and paraffin-embedded (FFPE) to allowfor histological analysis and for archival storage. Gene expressionlevels can be monitored semi-quantitatively and indirectly in such fixedand embedded samples by using immunohistochemical staining to monitorprotein expression levels. Because paraffin-embedded samples are widelyavailable, rapid and reliable methods are needed for the isolation ofnucleic acids, particularly RNA, from such samples.

A number of techniques exist for the purification of RNA from biologicalsamples, but none are reliable for isolation of RNA from FFPE samples.For example, Chomczynski (U.S. Pat. No. 5,346,994) describes a methodfor purifying RNA from tissues based on a liquid phase separation usingphenol and guanidine isothiocyanate. A biological sample is homogenizedin an aqueous solution of phenol and guanidine isothiocyanate and thehomogenate thereafter mixed with chloroform. Following centrifugation,the homogenate separates into an organic phase, an interphase and anaqueous phase. Proteins are sequestered in the organic phase, DNA in theinterphase, and RNA in the aqueous phase. RNA can be precipitated fromthe aqueous phase. This method does not provide for the reliableisolation of RNA from formalin-fixed paraffin-embedded tissue samples.

Other known techniques for isolating RNA typically utilize eitherguanidine salts or phenol extraction, as described for example inSambrook, J. et al., (1989) at pp. 7.3-7.24, and in Ausubel, F. M. etal., (1994) at pp. 4.0.3-4.4.7. However, none of the known methodsprovide reproducible quantitative results in the isolation of RNA fromparaffin-embedded tissue samples.

Techniques for the isolation of RNA from paraffin-embedded tissues areparticularly needed for the study of gene expression in tumor tissues.Expression levels of certain receptors or enzymes can indicate thelikelihood of success of a particular treatment.

Truly quantitative TS gene expression studies have been limited toRT-PCR from frozen tissue, whereas semi-quantitative monitoring of TSprotein expression in archival pathological material fixed onto glassslides has been available via immunohistochemical staining. Because oflimitations in isolating RNA from archival pathological material,quantitative techniques for measuring gene expression levels from suchsamples were heretofore unavailable.

SUMMARY

One aspect of the present invention is to provide a reliable method forthe isolation of RNA, DNA or proteins from samples of biologicaltissues. The invention also provides simple, efficient and reproduciblemethods for the isolation of RNA, DNA or proteins from tissue that hasbeen embedded in paraffin.

The invention provides methods of purifying RNA from a biological tissuesample by heating the sample for about 5 to about 120 minutes at atemperature of between about 50 and about 100° C. in a solution of aneffective concentration of a chaotropic agent. In one embodiment, thechaotropic agent is a guanidinium compound. RNA is then recovered fromsaid solution. For example, RNA recovery can be accomplished bychloroform extraction.

In a method of the invention, RNA is isolated from an archivalpathological sample. In one embodiment, a paraffin-embedded sample isfirst deparaffinized. An exemplary deparaffinization method involveswashing the paraffinized sample with an organic solvent, preferablyxylene. Deparaffinized samples can be rehydrated with an aqueoussolution of a lower alcohol. Suitable lower alcohols include, methanol,ethanol, propanols, and butanols. In one embodiment, deparaffinizedsamples are rehydrated with successive washes with lower alcoholicsolutions of decreasing concentration. In another embodiment, the sampleis simultaneously deparaffinized and rehydrated.

The deparaffinized samples can be homogenized using mechanical, sonic orother means of homogenization. In one embodiment, the rehydrated samplesare homogenized in a solution comprising a chaotropic agent, such asguanidinium thiocyanate (also sold as guanidinium isothiocyanate).

The homogenized samples are heated to a temperature in the range ofabout 50 to about 100° C. in a chaotropic solution, comprising aneffective amount of a chaotropic agent. In one embodiment, thechaotropic agent is a guanidinium compound. A preferred chaotropic agentis guanidinium thiocyanate.

RNA is then recovered from the solution by, for example, phenolchloroform extraction, ion exchange chromatography or size-exclusionchromatography.

RNA may then be further purified using the techniques of extraction,electrophoresis, chromatography, precipitation or other suitabletechniques.

RNA isolated by the methods of the invention is suitable for a number ofapplications in molecular biology including reverse transcription withrandom primers to provide cDNA libraries.

Purified RNA can be used to determine the level of gene expression in aformalin-fixed paraffin-embedded tissue sample by reverse transcription,polymerase chain reaction (RT-PCR) amplification. Using appropriate PCRprimers the expression level of any messenger RNA can be determined bythe methods of the invention. The quantitative RT-PCR technique allowsfor the comparison of protein expression levels in paraffin-embedded(via immunohistochemistry) with gene expression levels (using RT-PCR) inthe same sample.

The methods of the invention are applicable to a wide range of tissueand tumor types and target genes and so can be used for assessment oftreatment and as a diagnostic tool in a range of cancers includingbreast, head and neck, esophageal, colorectal, and others. Aparticularly preferred gene for the methods of the invention is thethymidylate synthase gene. The methods of the invention achievedreproducible quantification of TS gene expression in FFPE tissues,comparable to those derived from frozen tissue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows level of β-Actin and TS expression at various heatingtimes. These data show that without the heating step, there is a minimalyield of RNA extracted from the paraffin.

FIG. 2 shows the level of β-actin expression in normal (N) or tumorous(T) tissue from colorectal cancer patients as determined by quantitativePCR from RNA extracted at 95° C. for zero to 40 minutes. These datasuggest 30 min as an optimal heating time.

FIG. 3 shows the effect of both temperature and time on the yield ofβ-actin RNA and on the isolation of DNA. These data show that at longerheating times (between 60 and 120 min), RNA undergoes degradation whilethere is an increase in contaminating DNA capable of generating a DNAPCR signal. The bars represent values of triplicate experiments done atthe various times and temperatures indicated.

FIG. 4 shows the effect of various heating solutions on the yield ofisolated RNA. These data show that the chaotrope in the solution, inthis case guanidinium isothiocyanate (GITC), is the essential componentof the RNA extraction solution, without which the yield of extracted RNAis at least 10-fold lower.

FIG. 5 shows a comparison of relative TS gene expression fromparaffin-embedded (white bars) and frozen cell pellets (black bars) fromsix cell lines. These data show that analysis of paraffin-extracted RNAreliably reflects gene expression values in fresh-frozen tissue.

FIG. 6 shows a comparison of TS gene expression levels in normal ortumorous colon and tumorous esophageal tissues that were eitherformalin-fixed and paraffin-embedded or frozen.

FIG. 7 shows TS/β-actin ratios determined in paraffin sections frompatients whose response to 5-FU/LV was previously linked to TS geneexpression.

FIG. 8 shows the expression levels of four malignancy marker genes (TS;thymidine phosphorylase (TP); cyclooxygenase-2 (COX-2); and vascularendothelial growth factor (VEGF)) in FFPE samples of a primary coloncancer and a liver metastasis that recurred a year later in the samepatient. These data show that, as might be expected, three of the fourmalignancy markers are elevated in the metastatic tumor tissue.

DETAILED DESCRIPTION

The methods of the instant invention involve purification of RNA frombiological samples. In one embodiment, samples are paraffin-embeddedtissue samples and the method involves deparaffinization of embeddedsamples, homogenization of the deparaffinized tissue and heating of thehomogenized tissue at a temperature in the range of about 50 to about100° C. for a time period of between about 5 minutes to about 120minutes in a chaotropic solution containing an effective amount of aguanidinium compound. This heating step increases the amount of cDNAthat are amplified from the specimen by up to 1000-fold over samplesthat are not heated.

While frozen tumor tissue is not widely available, paraffin blocks areroutinely prepared from every type of tumor after surgery, allowinglarge-scale retrospective investigations of TS expression andchemotherapy response to be performed.

Moreover, the technique can be applied to any of a wide range of tumortypes and to an unlimited range of target genes. This has implicationsfor the future preparation of individual tumor “gene expressionprofiles” whereby expression levels could be determined in individualpatient samples for a range of genes that are known to influenceclinical outcome and response to various chemotherapeutic agents.Automated real-time PCR from FFPE sample allows for the targeting oftreatment to individual tumors.

Tissue Samples

RNA can be isolated from any biological sample using the methods of theinvention. Biological samples are often fixed with a fixative. Aldehydefixatives such as formalin (formaldehyde) and glutaraldehyde aretypically used. Tissue samples fixed using other fixation techniquessuch as alcohol immersion (Battifora and Kopinski, J. Histochem.Cytochem. (1986) 34:1095) are also suitable. The samples used are alsoembedded in paraffin. RNA can be isolated any paraffin-embeddedbiological tissue sample by the methods of the invention. In oneembodiment, the samples are both formalin-fixed and paraffin-embedded.

Deparaffinization of Samples

Deparaffinization removes the bulk of paraffin from theparaffin-embedded sample. A number of techniques for deparaffinizationare known and any suitable technique can be used with the presentinvention. The preferred method of the invention utilizes washing withan organic solvent to dissolve the paraffin. Such solvents are able toremove paraffin effectively from the tissue sample without adverselyaffecting RNA isolation. Suitable solvents can be chosen from solventssuch as benzene, toluene, ethylbenzene, xylenes, and mixtures thereof. Axylene is the preferred solvent for use in the methods of the invention.Solvents alone or in combination in the methods of the invention arepreferably of high purity, usually greater than 99%.

Paraffin is typically removed by washing with an organic solvent, withvigorous mixing followed by centrifugation. Samples are centrifuged at aspeed sufficient to cause the tissue to pellet in the tube, usually atabout 10,000 to about 20,000×g. After centrifugation, the organicsolvent supernatant is discarded. One of skill in the art of histologywill recognize that the volume of organic solvent used and the number ofwashes necessary will depend on the size of the sample and the amount ofparaffin to be removed. The more paraffin to be removed, the more washesthat will be necessary. Typically, a sample will be washed between 1 andabout 10 times, and preferably, between about two and about four times.A typical volume of organic solvent is about 500 μL for a 10 μm tissuespecimen.

Other methods for deparaffinization known to one of skill in the art mayalso be used in the method of the invention. Such methods include directmelting (Banerjee et al., 1995).

Samples are preferably rehydrated after deparaffinization. The preferredmethod for rehydration is step-wise washing with aqueous lower alcoholicsolutions of decreasing concentration. Ethanol is a preferred loweralcohol for rehydration. Other alcohols may also be suitable for usewith the invention including methanol, isopropanol and other similaralcohols in the C1-C5 range. The sample is alternatively vigorouslymixed with alcoholic solutions and centrifuged. In a preferredembodiment, the concentration range of alcohol is decreased stepwisefrom about 100% to about 70% in water over about three to fiveincremental steps, where the change in solution concentration at eachstep is usually less than about 10% (i.e., the sequence: 100%, 95%, 90%,80%, 70%). In another embodiment, deparaffinization and rehydration arecarried out simultaneously using a reagent such as EZ-DEWAX (BioGenex,San Ramon, Calif.).

Homogenization

Deparaffinized, rehydrated samples can be homogenized by any standardmechanical, sonic or other suitable technique. Tissue homogenization ispreferably carried out with a mechanical tissue homogenizers accordingto standard procedures. A number of commercially available homogenizersare suitable for use with the invention including: Ultra-Turraxhomogenizer (IKA-Works, Inc., Wilmington, N.C.); Tissumizer(Tekmar-Dohrmann, Cincinnati, Ohio); and Polytron (Brinkmann, Westbury,N.Y.).

In one embodiment, the sample is homogenized in the presence of achaotropic agent. Chaotropic agents are chosen such that at an effectiveconcentration RNA is purified from a paraffin-embedded sample in anamount of greater than about 10 fold that isolated in the absence of achaotropic agent. Chaotropic agents include: guanidinium compounds,urea, formamide, potassium iodiode, potassium thiocyantate and similarcompounds. The preferred chaotropic agent for the methods of theinvention is a guanidinium compound, such as guanidinium isothiocyanate(also sold as guanidinium thiocyanate) and guanidinium hydrochloride.Many anionic counterions are useful, and one of skill in the art canprepare many guanidinium salts with such appropriate anions. Theguanidinium solution used in the invention generally has a concentrationin the range of about 1 to about 5M with a preferred value of about 4M.If RNA is already in solution, the guanidinium solution may be of higherconcentration such that the final concentration achieved in the sampleis in the range of about 1 to about 5M. The guanidinium solution also ispreferably buffered to a pH of about 3 to about 6, more preferably about4, with a suitable biochemical buffer such as Tris-Cl. The chaotropicsolution may also contain reducing agents, such as dithiothreitol (DTT)and β-mercaptoethanol (BME). The chaotropic solution may also containRNAse inhibitors.

Heating

Samples are heated in the chaotropic solution at a temperature of about60° C. to about 100° C. for about 5 minutes to about 2 hours.Alternatively, samples are heated in the chaotropic solution at atemperature of about 50° C. to about 100° C. for about 5 minutes toabout 2 hours. Heating times are typically chosen such that the amountof RNA purified is at least about 100-fold higher than for unheatedsamples, and more preferably about 1000-fold higher. In a preferredembodiment, the sample is heated for about 20 minutes at a temperatureof from about 75 to about 100° C. More preferably, the sample is heatedfor 30 to 60 minutes at about 95° C.

RNA Recovery

RNA can be recovered from the chaotropic solution after heating by anysuitable technique that results in isolation of the RNA from at leastone component of the chaotropic solution. RNA can be recovered from thechaotropic solution by extraction with an organic solvent, chloroformextraction, phenol-chloroform extraction, precipitation with ethanol,isopropanol or any other lower alcohol, by chromatography including ionexchange chromatography, size exclusion chromatography, silica gelchromatography and reversed phase chromatography, or by electrophoreticmethods, including polyacrylamide gel electrophoresis and agarose gelelectrophoresis, as will be apparent to one of skill in the art. RNA ispreferably recovered from the chaotropic solution using phenolchloroform extraction.

Following RNA recovery, the RNA may optionally by further purified.Further purification results in RNA that is substantially free fromcontaminating DNA or proteins. Further purification may be accomplishedby any of the aforementioned techniques for RNA recovery. RNA ispreferably purified by precipitation using a lower alcohol, especiallywith ethanol or with isopropanol. Precipitation is preferably carriedout in the presence of a carrier such as glycogen that facilitatesprecipitation.

DNA and Protein Recovery

The methods of the invention can also be used to purify DNA or proteinfrom the tissue sample. After mixing a sample with an organic solvent,such as chloroform, and following centrifugation, the solution has threephases, a lower organic phase, an interphase, and an upper aqueousphase. With an appropriate chaotropic agent, particularly with aguanidinium compound, the biological components of the sample willsegregate into the three phases. The upper aqueous phase will containRNA, while the interphase will contain DNA and the organic phase willcontain proteins. One of skill in the art will recognize that themethods of the invention are suitable for recovering both the DNA andprotein phases as well as that containing the RNA. DNA recovery isenhanced by the methods of the invention.

Purified RNA

RNA purified by the methods of the invention is suitable for a varietyof purposes and molecular biology procedures including, but not limitedto: reverse transcription to cDNA; producing radioactively,fluorescently or otherwise labeled cDNA for analysis on gene chips,oligonucleotide microarrays and the like; electrophoresis by acrylamideor agarose gel electrophoresis; purification by chromatography (e.g. ionexchange, silica gel, reversed phase, or size exclusion chromatography);hybridization with nucleic acid probes; and fragmentation by mechanical,sonic or other means.

EXAMPLES Materials and Methods

These materials and methods are common to the following examples.

Sample Preparation. The characteristics of the human cell lines SK1,H157, A431, HT29, HCC298 and HH30 have been described previously. Cellswere removed from their monolayer by trypsinization and pelleted bycentrifugation at 3000 rpm for 5 minutes. Cell pellets were eitherfrozen at −70° C. or fixed in formalin for 24 h, then embedded inparaffin.

Representative sections of tumor tissue were obtained at the time ofsurgery, fixed in formalin and embedded in paraffin by procedures commonto most clinical pathology laboratories. Cross-sections of tissue (5 μm)were cut using a microtome.

RNA Isolation. RNA was isolated from paraffin embedded tissue asfollows. A single 5 μm section of paraffinized tissue was placed in anEppendorf tube and deparaffinized by two 15 minute washes with xylene.The tissue was rehydrated by successive 15 minute washes with gradedalcohols (100%, 95%, 80% and 70%). The resulting pellet was suspended in4M guanidine isothiocyanate with 0.5% sarcosine and 20 mM dithiothreitol(DTT). The suspension was homogenized and then heated to from about 50to about 95° C. for 0 to 60 minutes; a zero heating time-point, wasincluded as a control for each sample. Sodium acetate (pH 4.0) was addedto 0.2 M and the solution was extracted with phenol/chloroform andprecipitated with isopropanol and 10 mg glycogen. After centrifugation(13000 rpm, 4° C., 15 min) the RNA pellet was washed twice with 1 mL of75% ethanol then resuspended in RNase-free water.

Reverse transcription (RT). After heating, total RNA was converted tocDNA using random hexamers. RT conditions were as have been previouslydescribed for frozen tissue (Horikoshi et al., 1992). Controls omittingthe reverse transcriptase (No-RT) were prepared for each sample.

Real-Time PCR quantification of TS and β-actin gene expression using thePerkin Elmer Cetus 7700 PCR Machine (Taqman). The quantitation of mRNAlevels was carried out using real-time PCR based on a fluorescencedetection method as described previously (Heid et al., 1996; Eads etal., 1999). cDNA was prepared as described above. The cDNA of interestand the reference cDNA were amplified separately using a probe with a5′-fluorescent reporter dye (6FAM) and a 3′-quencher dye (TAMRA). The5′-exonuclease activity of TAQ polymerase cleaves the probe and releasesthe reporter molecule, the fluorescence of which is detected by the ABIPrism Sequence Detection System (Taqman). After crossing a fluorescencedetection threshold, the PCR amplification results in a fluorescentsignal proportional to the amount of PCR product generated. Initialtemplate concentration was determined from the cycle number at which thefluorescent signal crossed a threshold in the exponential phase of thePCR reaction. Relative gene expression was determined based on thethreshold cycles of the gene of interest and the reference gene. Use ofa reference gene avoids the need to quantitate the RNA directly, whichcould be a major source of error.

The primer and probe sequences were as follows: TS: SEQ ID NO: 1: GGCCTC GGT GTG CCT TT; SEQ ID NO:2: AAC ATC GCC AGC TAC GCC CTG C; SEQ IDNO:3: GAT GTG CGC AAT CAT GTA CGT. β-actin: SEQ ID NO:4: TGA GCG CGG CTACAG CTT; SEQ ID NO:5: ACC ACC ACG GCC GAG CGG; SEQ ID NO:6: TCC TTA ATGTCA CGC ACG ATT T. For the real-time PCR experiments, as discussedabove, the reporter oligonucleotide (SEQ ID NOS: 2 and 5) were 5′labelled with 6FAM and were 3′ labelled with TAMRA.

For each PCR, a “No Reverse Transcriptase” (NRT or No-RT) control wasincluded. The purpose of this reaction was to correct for any backgroundamplification, derived from residual genomic DNA contamination. Hence,each overall value for TS and β-actin is calculated as the RT valueminus the NRT value (RT-NRT).

Statistical Analysis. Non-parametric comparison of means test wereperformed to determine if differences in TS levels between frozen tissueand FFPE samples of the same tumor were significant or non-significant.

Example 1 General RNA Isolation Procedure

RNA was extracted from paraffin-embedded tissue by the following generalprocedure.

A. Deparaffinization and Hydration of Sections:

(1) A portion of an approximately 10 μM section is placed in a 1.5 mLplastic centrifuge tube.

(2) 600 μL of xylene are added and the mixture is shaken vigorously forabout 10 minutes at room temperature (roughly 20 to 25° C.).

(3) The sample is centrifuged for about 7 minutes at room temperature atthe maximum speed of the bench top centrifuge (about 10-20,000×g).

(4) Steps 2 and 3 are repeated until the majority of paraffin has beendissolved. Two or more times are normally required depending on theamount of paraffin included in the original sample portion.

(5) The xylene solution is removed by vigorously shaking with a loweralcohol, preferably with 100% ethanol (about 600 μL) for about 3minutes.

(6) The tube is centrifuged for about 7 minutes as in step (3). Thesupernatant is decanted and discarded. The pellet becomes white.

(7) Steps 5 and 6 are repeated with successively more dilute ethanolsolutions: first with about 95% ethanol, then with about 80% and finallywith about 70% ethanol.

(8) The sample is centrifuged for 7 minutes at room temperature as instep (3). The supernatant is discarded and the pellet is allowed to dryat room temperature for about 5 minutes.

B. RNA Isolation with Phenol-Chloroform

(1) 400 μL guanidine isothiocyanate solution including 0.5% sarcosineand 8 μL 1M dithiothreitol is added.

(2) The sample is then homogenized with a tissue homogenizer(Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to 3minutes while gradually increasing the speed from low speed (speed 1) tohigh speed (speed 5).

(3) The sample is then heated at about 95° C. for about 5-20 minutes. Itis preferable to pierce the cap of the tube containing the sample beforeheating with a fine gauge needle. Alternatively, the cap may be affixedwith a plastic clamp or with laboratory film.

(4) The sample is then extracted with 50 μL 2M sodium acetate at pH 4.0and 600 μL of phenolichloroform/isoamyl alcohol (10:1.93:0.036),prepared fresh by mixing 18 mL phenol with 3.6 mL of a 1:49 isoamylalcohol:chloroform solution. The solution is shaken vigorously for about10 seconds then cooled on ice for about 15 minutes.

(5) The solution is centrifuged for about 7 minutes at maximum speed.The upper (aqueous) phase is transferred to a new tube.

(6) The RNA is precipitated with about 10 μL glycogen and with 400 μLisopropanol for 30 minutes at −20° C.

(7) The RNA is pelleted by centrifugation for about 7 minutes in abenchtop centrifuge at maximum speed; the supernatant is decanted anddiscarded; and the pellet washed with approximately 500 μL of about 70to 75% ethanol.

(8) The sample is centrifuged again for 7 minutes at maximum speed. Thesupernatant is decanted and the pellet air dried. The pellet is thendissolved in an appropriate buffer for further experiments (e.g. 50 μL 5mM Tris chloride, pH 8.0).

Example 2 Heating Time

This example illustrates the effect of time of heating on the yield ofRNA.

As illustrated in FIG. 1, heating of the chaotropic solution at 95° C.prior to precipitation and reverse transcription significantly increasedthe efficiency of detection of TS and β-actin targets. When no heatingstep was included, neither TS nor β-actin could be detected (0 min. timepoints). After 20 minutes at 95° C. both transcripts were detectable; afurther increase of heating time to 60 minutes resulted in a 3-foldincrease in sensitivity of detection for TS and 4.5-fold increase forβ-actin. (NRT=No Reverse Transcriptase control, RT-NRT=overall relativegene expression level, i.e. Reverse Transcriptase minus No ReverseTranscriptase).

FIG. 2 illustrates the amount of RNA expression of the β-actin gene innormal (N) and tumorous (T) tissue. The samples were heated at 95° C.for periods ranging from zero to 40 minutes. A preferred heating time ofabout 30 minutes is observed for most samples.

FIG. 3 shows that at heating times longer than about 60 min, the amountof RNA extracted starts to decrease, suggesting thermal degradation ofthe RNA, whereas the amount of DNA extracted starts to increase. This isundesirable because the presence of DNA can give a spurious PCR signalin some cases.

Example 3 Heating Solutions

This example illustrates that heating the RNA solution in the presenceof a chaotropic agent is important for obtaining high yields of RNA.This was an RT-PCR experiment using detection of β-actin gene expressionas a measure of relative amounts of RNA isolated in various solutions.

Clinical specimens of esophageal cancer FFPE tissue samples were treatedaccording to the methods described above, with the exception that theinitial pellet obtained after deparaffinization was dissolved orsuspended in either 4M guanidinium isothiocyanate (GITC), 4M guanidiniumisothiocyanate+100 μM β-mercaptoethanol (GITC+BME), 4M guanidiniumisothiocyanate+20 μM dithiothreitol (GITC+DTT) or in Tris-Cl buffer (10mM pH 7.5) or Tris-Cl buffer+20 μM DTT (Tris/Cl+DTT). The samples werethen heated to 95° C. for 30 minutes or not heated (O min, 95° C.). TheTris/Cl samples were then treated with 4M guanidinium isothiocyanate.RNA levels were determined by RT-PCR and real time PCR detection ofβ-Actin. As shown in FIG. 4, the presence of the chaotropic agentguanidinium isothiocyanate when heating was important for high yieldrecovery of RNA. The presence of a reducing agent, such as DTT or BME,is not essential for high yield recovery of RNA. The 4M guanidiniumisothiocyanate solution contains 50 mM Tris-HCl (pH 7.5), 25 mM EDTA and0.5% Sarcosine.

Example 4 Comparison of Gene Expression Values Determined in FFPE andFrozen Tissue from the Same Sources

This example shows that the methods of the present invention providevalues for gene expressions from formalin-fixed paraffin-embeddedsamples equivalent to those obtained from frozen tissue.

Samples from six cell lines were FFPE-treated and TS quantitationperformed using the methods of the invention (including heating at 95°C. for 30 minutes). The resulting relative TS values (FIG. 5) werecompared with those obtained from frozen cell pellets using knownmethods. Relative TS expression levels were 3.0-19.5 (mean=8.5) infrozen cells versus 3.0-25.0 (mean=9.0) in FFPE samples. Statisticalanalysis of the difference between the two means revealed a p value of0.726, indicating that there is no significant difference in the TSvalues obtained from frozen cell pellets using the original RT-PCRmethods and those obtained from FFPE cell pellets using the methods ofthe invention.

RNA expression levels in samples of tumorous tissues and of normal(non-tumorous) tissues also were equivalent regardless of whether thesamples were formalin-fixed and paraffin-embedded or frozen. Five normaland 6 tumor colon tissues and 4 esophageal tumor tissues, were comparedfor relative TS gene expression in matching paraffin and frozen tissue(FT) as above. Results are illustrated in FIG. 6. No significantdifference was found between the levels of TS found in frozen tissuesamples and the TS values found in FFPE samples of the same tissue. Thiswas true for both colon and esophageal tissue types (mean FT samplescolon=3.46, mean FFPE samples colon=3.06, p=0.395; mean FT samplesesophagus=13.9, mean FFPE samples esophagus=15.93, p=0.21).

Example 5 Comparison of TS Levels in Responsive and Non-Responsive TumorTissues

Correlation of TS levels in frozen tissue and matching FFPE samples withresponse to 5-FU/Leucovorin (LV) in stage IV colon cancer. Previousreports based on RT-PCR data derived from frozen tissue found that highlevels of TS in tumors (relative gene expression ≧4.0) were indicativeof a poor response to TS treatment. Responsive tumors could becharacterized as expressing lower levels of TS. TS/β-actin ratios weredetermined in paraffin sections from 17 patients whose response to5-FU/LV had previously been linked to TS gene expression via analysis offrozen tissue samples (FIG. 7). Of the 17, 6 were known to be responsiveto TS and 11 were known to have been poor responders to TS treatment. Itwas found that the TS results with matching paraffin tissue would alsohave predicted response to this therapy (mean responders FT=2.87, meanresponders FFPE=2.37, p=0.641: mean non-responders FT=7.66, meannon-responders FFPE=7.84 p=0.537). There was no significant differencebetween the TS levels derived from frozen tissue and those derived frommatching FFPE tissues.

Example 6 TS Gene Expression Levels in Primary Colon Cancer and a LiverMetastasis

This example shows an analysis of TS, and other gene expression, in aprimary colon tumor and in a recurrent liver metastasis from the samepatient.

FIG. 8 shows the expression levels of four genes: TS; TP;cyclooxygenase-2 (COX-2); and vascular endothelial growth factor (VEGF)in FFPE samples of a primary colon cancer and a liver metastasis (met)from the same patient which recurred a year later. The findings suggestthat, while the primary tumor was sensitive to 5-FU therapy (TS=2.32),the metastasis will be refractory (TS met 11.58). COX-2 and VEGFexpression levels correlate with the published indications that they areincreased in expression in aggressive disease, and co-regulated. (Cox-2primary=1.35; COX-2 met=5.4; VEGF primary=5.02; VEGF met=14.4.) RNA wasisolated as described from a 5 μM FFPE section of the primary coloncancer and from an FFPE section of the liver metastasis. Relative TSgene expression in the responsive primary tumor was 2.32 compared to11.58 in the metastastic disease (FIG. 8). This 5-fold increase in TSexpression, as determined by the RT-PCR methods reported here, indicatesthat the secondary disease will not respond to 5-FU and suggests analternative therapy such as CPT-11 may be appropriate.

All references cited herein are hereby incorporated by reference intheir entirety.

REFERENCES

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1. A method for quantitative measurement of gene expression of a targetgene in a fixed paraffin embedded tissue sample, comprising: (a)deparaffinizing the sample to obtain a deparaffinized sample; (b)isolating RNA from the deparaffinized sample by heating thedeparaffinized sample in a chaotropic solution comprising an effectiveconcentration of a chaotropic agent to a temperature in the range ofabout 75 to about 100° C. for a time period of about 5 to about 120minutes and then recovering said RNA from said chaotropic solution toyield isolated RNA; (c) subjecting the isolated RNA to RT-PCRamplification using a pair of oligonucleotide primers capable ofamplifying a region of the target gene mRNA, to obtain an amplifiedsample; and (d) determining the quantity of target gene mRNA relative tothe quantity of an internal control gene's mRNA from the isolated mRNA,wherein the heating step increases the amount of cDNA that is amplifiedin the RT-PCR amplification step from 3 to 1000 fold in comparison to adeparaffinized tissue sample that is subject to similar RNA isolationconditions as in step (b) but not heated.
 2. The method of claim 1further comprising rehydrating the deparaffinized sample of step (b)before heating.
 3. The method of claim 1 wherein the isolated RNA isrecovered with a water insoluble organic solvent.
 4. The method of claim3 wherein said water insoluble organic solvent comprises chloroform. 5.The method of claim 1 wherein the isolated RNA is recovered from saidchaotropic solution by ethanol precipitation.
 6. The method of claim 1wherein said time period is from about 10 to about 60 minutes.
 7. Themethod of claim 6 wherein said time period is from about 30 to about 60minutes.
 8. The method of claim 1 wherein said temperature is in therange of about 85 to about 100° C.
 9. The method of claim 8 wherein saidtime period is from about 30 to about 60 minutes.
 10. The method ofclaim 1 wherein said chaotropic agent is a guanidinium compound.
 11. Themethod of claim 1 wherein said guanidinium compound is guanidiniumhydrochloride.
 12. The method of claim 1 wherein said guanidiniumcompound is guanidinium isothiocyanate.
 13. The method of claim 12wherein said guanidinium isothiocyanate is present in a concentration ofabout 2 to about 5 M.
 14. The method of claim 13 wherein saidguanidinium isothiocyanate is present in a concentration of about 4 M.15. The method of claim 10 wherein said chaotropic solution has a pH ofabout 3 to
 6. 16. The method of claim 10 wherein said chaotropicsolution has a pH of about
 4. 17. The method of claim 1 wherein saidchaotropic solution further comprises a reducing agent.
 18. The methodof claim 17 wherein said reducing agent is β-mercaptoethanol.
 19. Themethod of claim 17 wherein said reducing agent is dithiothreitol. 20.The method of claim 1, wherein the tissue sample is formalin-fixed andparaffin embedded (FFPE).
 21. The method of claim 1 wherein the relativequantity of target gene mRNA in said tissue sample is the same whethersaid tissue sample is formalin-fixed and paraffin embedded or frozen.22. The method of claim 1, wherein the internal control gene is β-actin.23. A method for determining the level of a target gene expression in afixed paraffin embedded tissue sample, comprising: (a) deparaffinizingthe sample to obtain a deparaffinized sample; (b) isolating RNA from thedeparaffinized sample by heating the deparaffinized sample in a solutioncomprising an effective concentration of a chaotropic agent to atemperature in the range of about 75 to about 100° C. and thenrecovering said RNA from said solution; and (c) determining the quantityof the target gene mRNA relative to the quantity of an internal controlgene's mRNA using RT-PCR, wherein the heating step increases the amountof cDNA that is amplified in the RT-PCR from 3 to 1000 fold incomparison to a deparaffinized tissue sample that is subject to similarRNA isolation conditions as in step (b) but not heated.
 24. The methodof claim 23, wherein the heating takes place from about 5 to about 120minutes.
 25. A method for quantitative measurement of gene expression ofa target gene in a fixed paraffin embedded tissue sample, comprising:(a) deparaffinizing the tissue sample to obtain a deparaffinized samplecomprising DNA and RNA; (b) rehydrating the deparaffinized sample; (c)isolating RNA from the rehydrated sample by heating the rehydratedsample in a solution comprising an effective concentration of achaotropic agent to a temperature in the range of about 75 to about 100°C. for a time period of about 5 to about 120 minutes and then recoveringsaid RNA from said chaotropic solution to yield isolated RNA; (d)subjecting the isolated RNA to RT-PCR amplification using a pair ofoligonucleotide primers capable of amplifying a region of the targetgene mRNA, to obtain an amplified sample; and (e) determining thequantity of target gene mRNA relative to the quantity of an internalcontrol gene's mRNA from the isolated mRNA, wherein the heating stepincreases the amount of cDNA that is amplified in the RT-PCRamplification step from 3 to 1000 fold in comparison to a rehydratedtissue sample that is subject to similar RNA isolation conditions as instep (c) but not heated.
 26. The method of claim 25 wherein recoveringthe isolated RNA comprises a recovery technique selected from the groupconsisting of extraction, electrophoresis, chromatography,precipitation, and a combination thereof.
 27. The method of claim 1wherein the RNA is isolated free of DNA present in the sample.
 28. Themethod of claim 25 wherein the RNA is isolated free of DNA present inthe sample.